Physiologie eines erlernten Körpermodells in Drosophila melanogaster

In der vorliegenden Dissertation wird ein Körpergrößengedächtnis untersucht. Es wird dargestellt, wie diese Information über die Reichweite der Fliege beim Lückenklettern unter kotrollierten Umweltbedingungen erworben und prozessiert wird. Zusätzlich wird geklärt, welche biochemischen Signale benötigt werden, um daraus ein lang anhalten-des Gedächtnis zu formen. Adulte Fliegen sind in der Lage, ihre Körperreichweite zu lernen. Naive Fliegen, die in der Dunkelheit gehalten wurden, versuchen erfolglos, zu breite Lücken zu überqueren, während visuell erfahrene Fliegen die Kletterversuche an ihre Körpergröße anpassen. Erfahrene kleine Fliegen scheinen Kenntnis ihres Nachteils zu haben. Sie kehren an Lückenbreiten um, welche ihre größeren Artgenos-sen durchaus noch versuchen. Die Taufliegen lernen die größenabhängige Reichweite über die visuelle Rückmeldung während des Laufens (aus Parallaxenbewegung). Da-bei reichen 15 min in strukturierter, heller Umgebung aus. Es gibt keinen festgelegten Beginn der sensiblen Phase. Nach 2 h ist das Gedächtnis jedoch konsolidiert und kann durch Stress nicht mehr zerstört oder durch sensorische Eingänge verändert werden. Dunkel aufgezogene Fliegen wurden ausgewählten Streifenmustern mit spezifischen Raumfrequenzen ausgesetzt. Nur die Insekten, welche mit einem als „optimal“ klassi-fizierten Muster visuell stimuliert wurden, sind in der Lage, die Körperreichweite einzu-schätzen, indem die durchschnittliche Schrittlänge in Verbindung mit der visuellen Wahrnehmung gebracht wird. Überraschenderweise ist es sogar mittels partieller Kompensation der Parallaxen möglich, naive Fliegen so zu trainieren, dass sie sich wie kleinere Exemplare verhalten. Da die Experimente ein Erlernen der Körperreich-weite vermuten lassen, wurden lernmutante Stämme beim Lückenüberwinden getes-tet. Sowohl die Ergebnisse von rut1- und dnc1-Mutanten, als auch das defizitäre Klet-tern von oc1-Fliegen ließ eine Beteiligung der cAMP-abhängigen Lernkaskade in der Protocerebralbrücke (PB) vermuten. Rettungsexperimente der rut1- und dnc1-Hinter-gründe kartierten das Gedächtnis in unterschiedliche Neuronengruppen der PB, wel-che auch für die visuelle Ausrichtung des Kletterns benötigt werden. Erstaunlicher-weise haben laterale lokale PB-Neurone und PFN-Neurone (Projektion von der PB über den fächerförmigen Körper zu den Noduli) verschiedene Erfordernisse für cAMP-Signale. Zusammenfassend weisen die Ergebnisse darauf hin, dass hohe Mengen an cAMP/PKA-Signalen in den latero-lateralen Elementen der PB benötigt werden, wäh-rend kolumnäre PFN-Neurone geringe oder keine Mengen an cAMP/PKA erfordern. Das Körperreichweitengedächtnis ist vermutlich das am längsten andauernde Ge-dächtnis in Drosophila. Wenn es erst einmal konsolidiert ist hält es länger als drei Wo-chen.rnAußerdem kann die Fruchtliege Drosophila melanogaster trainiert werden, die kom-plexe motorische Aufgabe des Lückenkletterns zu optimieren. Die trainierten Fliegen werden erfolgreicher und schneller beim Überqueren von Lücken, welche größer sind als sie selbst. Dabei existiert eine Kurzeitkomponente (STM), die 40 min nach dem ersten Training anhält. Nach weiteren vier Trainingsdurchläufen im Abstand von 20 min wird ein Langzeitgedächtnis (LTM) zum Folgetag geformt. Analysen mit Mutati-onslinien wiesen eine Beteiligung der cAMP-abhängigen Lernkaskade an dieser Ge-dächtnisform auf. Rettungsexperimente des rut2080-Hintergrunds kartierten sowohl das STM, als auch das LTM in PFN-Neuronen. Das STM kann aber ebenso in den alpha- und beta- Loben der Pilzkörper gerettet werden.rnLetztendlich sind wildtypische Fliegen sogar in der Lage, sich an einen Verlust eines Mittelbeintarsuses und dem einhergehenden Fehlen des Adhäsionsorgans am Tarsusende anzupassen. Das Klettern wird zwar sofort schlechter, erholt sich aber bis zum Folgetag wieder auf ein normales Niveau. Dieser neue Zustand erfordert ein Ge-dächtnis für die physischen Möglichkeiten, die nur durch plastische Veränderungen im Nervensystem des Insekts erreicht werden können.

Expression of adipokines and estrogen receptors in adipose tissue and placenta of patients with gestational diabetes mellitus

The purpose of this study was to assess the expression profile of genes with potential role in the development of insulin resistance (adipokines, cytokines/chemokines, estrogen receptors) in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placenta of pregnant women with gestational diabetes mellitus (GDM) and age-matched women with physiological pregnancy at the time of Caesarean section. qRT-PCR was used for expression analysis of the studied genes. Leptin gene expression in VAT of GDM group was significantly higher relative to control group. Gene expressions of interleukin-6 and interleukin-8 were significantly increased, whereas the expressions of genes for estrogen receptors alpha and beta were significantly reduced in SAT of GDM group relative to controls, respectively. We found no significant differences in the expression of any genes of interest (LEP, RETN, ADIPOR1, ADIPOR2, TNF-alpha, CD68, IL-6, IL-8, ER alpha, ER beta) in placentas of women with GDM relative to controls. We conclude that increased expression of leptin in visceral adipose depot together with increased expressions of proinflammatory cytokines and reduced expressions of estrogen receptors in subcutaneous fat may play a role in the etiopathogenesis of GDM.

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.

A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle

Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (-)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 10(3) times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current I(Ca(L)) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. alpha- and beta-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 microM, 0.36 mg kg(-1), respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.

Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD). SAMPLE POPULATION: Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter). PROCEDURES: Concentrations of mRNA for 9 adrenoceptor subtypes (alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), beta(1), beta(2), and beta(3)) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA for alpha(1B)-, alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors, and in the cecum and PLAC for alpha(2B)- and beta(2)-adrenoceptors. Groups did not differ significantly for alpha(1A)-adrenoceptors. The mRNA expression for alpha(1D)-, alpha(2C)-, and beta(3)-adrenoceptors was extremely low in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Impact of subunit positioning on GABAA receptor function

The major isoforms of the GABAA (gamma-aminobutyric acid type A) receptor are composed of two alpha, two beta and one gamma subunit. Thus alpha and beta subunits occur twice in the receptor pentamer. As it is well documented that different isoforms of alpha and beta subunits can co-exist in the same pentamer, the question is raised whether the relative position of a subunit isoform affects the functional properties of the receptor. We have used subunit concatenation to engineer receptors of well-defined subunit arrangement to study this question. Although all five subunits may be concatenated, we have focused on the combination of triple and dual subunit constructs. We review here what is known so far on receptors containing simultaneously alpha1 and alpha6 subunits and receptors containing beta1 and beta2 subunits. Subunit concatenation may not only be used to study receptors containing two different subunit isoforms, but also to introduce a point mutation into a defined position in receptors containing either two alpha or beta subunits, or to study the receptor architecture of receptors containing unconventional GABAA receptor subunits. Similar approaches may be used to characterize other members of the pentameric ligand-gated ion channel family, including nicotinic acetylcholine receptors, glycine receptors and 5-HT3 (5-hydroxytryptamine) receptors.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate cross-reactivity to related compounds

In the context of drug hypersensitivity, our group has recently proposed a new model based on the structural features of drugs (pharmacological interaction with immune receptors; p-i concept) to explain their recognition by T cells. According to this concept, even chemically inert drugs can stimulate T cells because certain drugs interact in a direct way with T-cell receptors (TCR) and possibly major histocompatibility complex molecules without the need for metabolism and covalent binding to a carrier. In this study, we investigated whether mouse T-cell hybridomas transfected with drug-specific human TCR can be used as an alternative to drug-specific T-cell clones (TCC). Indeed, they behaved like TCC and, in accordance with the p-i concept, the TCR recognize their specific drugs in a direct, processing-independent, and dose-dependent way. The presence of antigen-presenting cells was a prerequisite for interleukin-2 production by the TCR-transfected cells. The analysis of cross-reactivity confirmed the fine specificity of the TCR and also showed that TCR transfectants might provide a tool to evaluate the potential of new drugs to cause hypersensitivity due to cross-reactivity. Recombining the alpha- and beta-chains of sulfanilamide- and quinolone-specific TCR abrogated drug reactivity, suggesting that both original alpha- and beta-chains were involved in drug binding. The TCR-transfected hybridoma system showed that the recognition of two important classes of drugs (sulfanilamides and quinolones) by TCR occurred according to the p-i concept and provides an interesting tool to study drug-TCR interactions and their biological consequences and to evaluate the cross-reactivity potential of new drugs of the same class.

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.

Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

Snake venom C-type lectins interacting with platelet receptors. Structure-function relationships and effects on haemostasis

Snake venoms contain components that affect the prey either by neurotoxic or haemorrhagic effects. The latter category affect haemostasis either by inhibiting or activating platelets or coagulation factors. They fall into several types based upon structure and mode of action. A major class is the snake C-type lectins or C-type lectin-like family which shows a typical folding like that in classic C-type lectins such as the selectins and mannose-binding proteins. Those in snake venoms are mostly based on a heterodimeric structure with two subunits alpha and beta, which are often oligomerized to form larger molecules. Simple heterodimeric members of this family have been shown to inhibit platelet functions by binding to GPIb but others activate platelets via the same receptor. Some that act via GPIb do so by inducing von Willebrand factor to bind to it. Another series of snake C-type lectins activate platelets by binding to GPVI while yet another series uses the integrin alpha(2)beta(1) to affect platelet function. The structure of more and more of these C-type lectins have now been, and are being, determined, often together with their ligands, casting light on binding sites and mechanisms. In addition, it is relatively easy to model the structure of the C-type lectins if the primary structure is known. These studies have shown that these proteins are quite a complex group, often with more than one platelet receptor as ligand and although superficially some appear to act as inhibitors, in fact most function by inducing thrombocytopenia by various routes. The relationship between structure and function in this group of venom proteins will be discussed.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLC gamma 2. Mucetin-induced phosphorylation of the Fc gamma chain of platelet was greatly promoted by inhibition of alpha(IIb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of alpha(IIb)beta(3) activation are involved in dephosphorylation of Fc gamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(IIb)beta(3). Inhibition of alpha(IIb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A2 are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor alpha IIb beta 3, has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of alpha IIb beta 3 inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.